The present invention relates to a composition for the preservation of organs intended for transplantation and a method of preserving organs intended for transplantation using the composition.
Transplantation of organs is now considered to be a definitive treatment for patients with end stage liver, kidney, heart and pancreas disease. There is thus a great deal of interest in improving ex vivo storage of cadaveric organs and thus the viability of organ transplants.
The two most commonly used methods for organ preservation are hypothermic storage and continuous pulsatile perfusion. With hypothermic storage, the organs are rapidly cooled immediately after removal from the cadaver donor using a combination of external cooling and a short period of perfusion. The hypothermic storage method is a preferred method due to its practicality and the ease of transportation of the organs. Continuous pulsatile perfusion involves hypothermic pulsatile perfusion after flushing with a chilled electrolyte solution.
A number of organ preservation solutions have been developed with a view to extending organ preservation time. Ringer's lactate and isotonic saline solutions have been used as extracellular flushing solutions and have been reported to allow for safe renal preservation for short periods of time, i.e. up to four hours. Storage for longer periods of time may result in severe histologic ischemic damage and subsequent non-function of the organs (column 5, U.S. Pat. No. 4,920,004)
An intracellular electrolyte solution developed by Collins et al., (Lancet 2:1219, 1969), has been reported to offer several advantages for hypothermic storage. Table I shows the composition of the Collins solution which is most commonly used. Modified Collins' solutions have also been developed for use in hypothermic storage. For example, Euro-collins solution is similar to Collins solution with the exception that it does not contain magnesium.
Other organ preservation solutions which have been developed include Sacks' solutions (S.sub.1 and S.sub.2) which have high intracellular ion concentration and osmotic pressure (Sacks, S. A., Lancet 1:1024, 1973). Table I shows the composition of the Sacks-2 flush solution. The solutions have been reported to provide improved transplantation results after storage of kidneys for up to 72 hours.
Protective additives such as ATP-MgCl.sub.2, AMP-Mg-Cl.sub.2 and inosine have also been included in preservation/flush solutions. (Siegel, N. J. et al., Am. J. Physiol. 245:F530, 1983: Stromski, M. E. et al, Am. J. Physiol. 250:F834, 1986; Sumpio, B. E. et al., Am. J. Physiol. 247:R1047; Stromski, M. E. et al., Am J. Physiol, 250:F834, 1986). Belzer et al., (Transpl. Proc. 16:161, 1984) developed a perfusate containing ATP-MgCl.sub.2 (see Table I for ingredients) but it has not been used for simple cold storage (Arch. Surg. 122:790-794, 1987).
U.S. Pat. No. 4,920,004 discloses a hyperosmotic intracellular flush and storage solution that is reported to combine the salient features of Belzer's ATP-MgCl.sub.2 perfusat commonly used Collins' C-2 Flush solutions. Mannitol is substituted in place of dextrose in Collins. C-2 solution and adenosine and magnesium are added to the solution to improve the preservation properties of the flush solution.
U.S. Pat. Nos. 4,798,824 and 4,873,230 disclose solutions for the preservation of organs (particularly kidneys) prior to implantation, containing a specific synthetic hydroxyethyl starch in place of serum albumin to produce the required oncotic pressure. In particular, U.S. Pat. No. 4,798,824 discloses a solution including 5% hydroxyethyl starch having a molecular weight of from about 200,000 to about 300,000 and wherein the hydroxyethyl starch is substantially free of ethylene glycol, ethylene chlorohydrin, sodium chloride and acetone. U.S. Pat. No. 4,873,230 discloses a solution containing hydroxyethyl starch having a molecular weight of from about 150,000 to about 350,000 daltons, a degree of substitution of from about 0.4 to about 0.7 and being substantially free of ethylene glycol, ethylene chlorohydrin, sodium chloride and acetone.
Marshall's isotonic citrate solution (Table I) has been reported to be capable of prolonging the period of safe hypothermic organ storage. It contains the impermeant anion, citrate and other substances which are believed to prevent free radical injury.
U.S. Pat. No. 4,879,283, discloses a solution for the preservation of organs which contains lactobionate and raffinose and has a solution osmolality of about 320 mOsm/L, K.sup.+ of 120 mM and Na.sup.+ of 20 mM. The solution also contains a synthetic hydroxyethyl starch and other components such as glutathione and adenosine. The solution disclosed in U.S. Pat. No. 4,879,283 is commonly known as the University of Wisconsin solution or UW solution and its composition is shown in Table I. The solution has been reported to successfully preserve the liver (Jamieson, N. V. et al., Transplantation 46:517, 1988), kidney (Ploeg, R.J. et al., Transplantation 46:191, 1988), and pancreas (Wahlberg, J. A., Transplantation, 43:5, 1987). Preliminary animal studies suggest that the solution may also be effective for the preservation of the heart (Wicomb, W. N., Transplantation 47:733, 1988; and Swanson, D. K., J. Heart Transplant 7:456, 1988).
The UW solution and Marshall's isotonic citrate solution are believed to provide improved organ preservation as a result of their ability to prevent cell swelling or oxygen free radical-mediated injury (Belzer, F. O. and Southard, J. H., In: Transplantation: Approaches to Graft Rejection. N.Y.,: List, 291, 1986; Toledo-Pereyra, L. H. et al, Ann. Surg. 181:289, 1975; Downes, G. et al, Transplantation 16:46, 1973; and Green, G. S. Pegg, D. E., In: Pegg, D. E., Jacobson, J. A., eds. Organ preservation. Edinburgh: Churchill Livingstone, 86, 1979). The solutions contain impermeant anions, citrate or lactobionate, which are added to maintain the normal double-Donnan equilibrium and prevent cell swelling in spite of the inactivation of the Na/K ATPase - dependent pump by hypothermia (Belzer, F. O. and Southard, J. H., In: Transplantation: Approaches to Graft Rejection. N.Y.: Liss, 291, 1986; Martin D. R. et al, Ann Surg 175:111, 1972; Southard, J. H. and Belzer, F. O. Cryobiology 17:540, 1980; and Mees N. et al., J. Trauma 22:118, 1982).
The present inventors have studied the relationship between transplant viability and liver function. In particular, the present inventors using a rat liver model have found that AST (aspartate aminotransferase) and LDH (lactate dehydrogenase) concentration in perfusate, discriminated between viable and nonviable livers across as well as within preservation groups. AST was found to give the best separation between viable and nonviable livers. Functions such as ALT (alanine aminotransferase) concentration in perfusate were found to separate viable from nonviable liver allografts only within preservation groups. In studying markers of allograft viability, the present inventors observed that rat livers stored at 1.degree. C. for 4 hours or at 37.degree. C. for 1 hour in a simple preservation solution (NaCl 0.9%, CaCl.sub.2, 2 mM) were all viable on transplantation but those stored at 4.degree. C. for 8 hours or at 37.degree. C. for 2 hours were nonviable. Cold preserved nonviable livers were also shown to have increased vascular resistance, platelet trapping and an initially low, but then high rise in AST upon reperfusion, all suggesting injury to the microcirculation. (Iu, S. et al, Transplantation, 44:562, 1987).
The present inventors have further identified the morphological changes that occur in livers stored for the above-mentioned critical times using light and electron microscopy after perfusion fixation. (McKeown, C. M. B. et al., Transplantation, 46:178, 1988). The present inventors observed that reversible injury was manifest by partial disruption of the endothelium and swelling of sinusoidal lining cells; hepatocytes appeared essentially normal, apart from some minor bleb formation. Irreversible injury and loss of viability was characterized by a completely deficient endothelium; lining cells were round with dark nuclei, and were detached from the underlying hepatocytes.
The present inventors also compared the effects on the microcirculation of preservation in UW and Marshall's solution and other control solutions and concluded that injury to the microcirculation was due neither to free-radical-mediated injury nor to cell swelling (Holloway, C.M.B. et al., 48, 179, 1989).